Lipoxygenase Involvement in Ripening Strawberry ANTONELLA LEONE,* ,† TERESA BLEVE-ZACHEO, ‡ CARMELA GERARDI, † MARIA T. MELILLO, ‡ LUCIA LEO, † AND GIUSEPPE ZACHEO † Consiglio Nazionale delle Ricerche, Istituto di Scienze delle Produzioni Alimentari, 73100 Lecce, Italy, and Consiglio Nazionale delle Ricerche, Istituto per la Protezione delle Piante, 70126 Bari, Italy The enzymatic activity, subcellular localization, and immunolocalization of plant lipoxygenase (LOX) in strawberry fruits (Fragaria × ananassa, Duch) were investigated. Chemical and enzymatic properties of LOX have been characterized, and the LOX capability of oxygenating free and esterified unsaturated fatty acids into C 6 volatile aldehydes has been confirmed. Fruits at unripe, turning, and ripe stages were analyzed for LOX activity and protein localization by Western blots, two-dimensional electro- phoresis, and immunolocalization analyses. The ability of strawberry tissues to in vivo metabolize linolenic acid or linoleic acid into C 6 volatile aldehydes and the LOX products was also analyzed. Analysis of strawberry proteins showed that a number of LOX forms, corresponding to at least two mobility groups of approximately 100 and 98 kDa and pI values ranging between 4.4 and 6.5, were present. Confocal and electron microscopy analyses support the idea that LOX proteins are associated to lipid-protein aggregates. Both exogenously supplied linoleate and linolenate were converted into hexanal and trans-2-hexenal at the three fruit-ripening stages. Our experiments suggest the presence of different LOX isoforms in strawberry fruits and that the lipoxygenase-hydroperoxide lyase pathway plays a role in converting lipids to C 6 volatiles during ripening. KEYWORDS: Strawberry (Fragaria × ananassa, Duch); C6 aldehydes; polyunsaturated fatty acids; lipoxygenase (LOX); hydroperoxide lyase (HPL); hydroperoxides (HPO) INTRODUCTION Lipoxygenases (LOXs; linoleate, oxygen oxidoreductase, E.C.1.13.11.12) are a family of nonheme, iron-containing dioxygenases, widely distributed in plants and animals. Several LOX isoforms have been identified in different cell compart- ments (1-4), and their activity has been associated to mem- branes of different origin, such as chloroplasts (5), lipid bodies (6), tonoplasts (7), and isolated plasma membranes (8). Chemical and enzymatic properties of plant LOXs have been extensively characterized, and it has been widely accepted that plant LOXs have various physiological roles in the plant metabolism (2). The presence of a number of LOX isoforms in the different subcellular compartments makes it difficult to assign a specific function to each LOX isoform. Some evidence suggests that LOXs are correlated with certain processes: growth regulation, maturation, senescence, and wound- and pathogen-induced defense (9-12). Plant LOXs catalyze the dioxygenation of 1,4 pentadiene cis- polyunsaturated fatty acids into their corresponding hydroper- oxide derivatives (HPO), highly reactive compounds, which can be further metabolized by other enzymes of the so-called LOX pathway [allene oxide synthase, hydroperoxide lyase (HPL), peroxygenase, or divinyl ether synthase] into a cascade of bioactive products (2). The oxidation of lipids, by the sequential action of LOX and HPL, is reported to produce volatile compounds, which contribute to flavor and aroma in many plant tissues (13-16). Distinct plant LOX isozymes preferentially incorporate oxygen into either the C-9 or the C-13 position of linoleic (LA) and linolenic acids (LNA) to produce 9-(S)- or 13-(S)-hydroperoxy octadecadienoic acid (HPOD) or 9-(S)- or 13-(S)-hydroperoxy octadecatrienoic acid (HPOT), respectively. Both 9- and 13-hydroperoxides can subsequently be cleaved to C 12 or C 9 ω-oxo-fatty-acids and volatile C 6 and C 9 aldehydes, respectively, by the action of HPL enzymes, recently character- ized as a special class of cytochrome P450 (17). Volatile aliphatic C 6 compounds are important constituents of charac- teristic fruit flavors and aromas, especially of the “fresh green” aroma. The involvement of the LOX pathway in the biosynthesis of fruit aroma compounds has been reported in tomato (18, 19), olive (15), bell pepper (20), apple (14), citrus (21), and strawberry (13). The function of LOXs and HPL in fruit ripening is not known, but their involvement in strawberry ripening has been suggested (13). The development of aroma in strawberry fruits is a very complex process involving a large number of volatile molecules such as esters, saturated and unsaturated aldehydes and alcohols, sulfur compounds, and furanone-derived compounds (22, 23). The components of strawberry aroma are well-known (24), and * To whom correspondence should be addressed. Tel: +39 0832 422615. Fax: +39 0832 422620. E-mail: antonella.leone@ispa.cnr.it. † Istituto di Scienze delle Produzioni Alimentari. ‡ Istituto per la Protezione delle Piante. J. Agric. Food Chem. 2006, 54, 6835-6844 6835 10.1021/jf061457g CCC: $33.50 © 2006 American Chemical Society Published on Web 08/08/2006